EGF binds to cell surface receptors (EGF-R) to stimulate their intrinsic tyrosine kinase activity and initiate a signal transduction cascade that regulates cell growth and differentiation. After ligand binding, occupied EGF-R are concentrated in coated pits and rapidly internalized via clathrin coated vesicles. The role of endocytosis of polypeptide growth factors and their receptors has not been established but it is thought to be critical for the attenuation of their cell proliferative signals. This proposal takes advantage of two important technological advances recently made in the laboratory. First, the investigators have established stable human cell lines which exhibit specific and conditional defects in clathrin-dependent receptor-mediated endocytosis providing an unprecedented opportunity to directly assess the role of endocytosis in regulating the proliferative responses of cells to EGF and other peptide growth factors. Thus, early and late events in the EGF-triggered signal transduction cascade will be measured in cells expressing endogenous EGF-receptors and compared under conditions permissive and non-ermissive for endocytosis. Second, they have established a unique cell-free assay for ligand-induced, kinase-dependent recruitment of activated EGF-R into coated pits and shown that a kinase substrate(s) distinct from the EGF-R itself, is required for efficient ligand induced endocytosis. This functional assay provides the means to identify and isolate the tyrosine kinase substrate(s) required for the first step in receptor down-regulation, namely the efficient ligand-induced recruitment of activated EGF-R into coated pits. A direct test for the role of this factor in vivo will be performed by expressing the cloned protein in stably transformed cells and characterizing its effect on EGF-R endocytosis and on regulating cellular responses to EGF. Understanding the physiological role of rapid EGF-R endocytosis in regulating cell proliferative responses will be important for therapeutically manipulating pathogenic, unregulated cell proliferation in breast (and other) cancers.